Confocal microscopes for scanning tissue can produce microscopic image sections of tissue. Such microscopic image sections may be made in-vivo in tissue without requiring a biopsy specimen of the lesion. Examples of confocal scanning microscopes are found in Milind Rajadhyaksha et al., “In-vivo Confocal Scanning Laser Microscopy of Human Skin: Melanin provides strong contrast,” Journal of Investigative Dermatology, Volume 104, No. 6 June 1995, pages 1-7, and in Milind Rajadhyaksha et al., “Confocal laser microscope images tissue in vivo,” Laser Focus World, February 1997 pages 119-127. These systems have confocal optics which direct light to the patient's tissue and image the returned reflected light. Imaging in the confocal microscope may be with illumination at different or multiple wavelengths which may be applied simultaneously. Microscope images of tissue sections can also be produced by optical coherence tomography or interferometry, such as described in Schmitt et al., “Optical characterization of disease tissues using low-coherence interferometry,” Proc. Of SPIE, volume 1889 (1993). Another type of imager used to image microscopic section is a two-photon laser microscope, such as described in U.S. Pat. No. 5,034,613 to Denk et al., issued Jul. 23, 1991. Finally, microscopic images sections can be imaged using high-frequency ultra-sound imaging systems such as DermaScanC manufactured by Cortex Technologies of Denmark.
Confocal microscopes and optical coherence tomography systems image native optical refractive index variation within the epithelial and stromal compartments of the tissue. These refractive index variations are due to the chemical variations and structural configurations of the chemical compounds within the tissue. Structures that backscatter more light appear brighter than less scattering structures. In general, whenever there is a large refractive index change between two cellular structures, there is the possibility for a high degree of backscatter. Ultrasound systems image the native sonic refractive index variations within the epithelial and stromal compartments of the tissue. These sonic refractive index variations are related to the density variations in the tissue. Two-photon microscopy images the fluorescence spectra of the tissue components.
Traditional pathological interpretation of cellular images focuses on the identification of cell type and its location and the morphology of the cellular structures for example the nuclear to cytoplasm ratio. Thus, it is important that the images of in-vivo and ex-vivo tissue have contrast sufficient to allow for the visualization of the overall cell size and the geometry of the nucleus.
Depending on the tissue type and its condition, different cellular components image with different contrast. For example, the nuclei of oral mucosal cells image appear bright compared to its cytoplasm, when viewed by near-infrared confocal microscopy. The nuclei of granular cells in the skin image dark compared to the cytoplasm of the skin when viewed by near-infrared confocal microscopy. The difference can be attributed to the difference in refractive index between the cytoplasm and nuclei, and the size and shape of the structures that make up the nucleus and cytoplasm. The nuclei of both oral mucosa and granular cells contain nucleic acids. The cytoplasm of oral mucosal cells is highly aqueous which suspend direct organelles such as mitochondria. The average refractive index of the cytoplasm is thus close to a normal saline solution approximately 1.34. The cytoplasm of granular cells is filled with the keratohyalin granules. The granules have a refractive index approaching 1.5. Their size, which is on the order of a wavelength of light, also facilitates the backscatter of light.
Thus, it is desirable to provide for a method and solution to modify the configuration of the constituents of in-vivo and ex-vivo tissues so as to enhance the visibility of cellular structures, such as the nucleus of the cell, which may use staining agents other than acetic acid solutions as have heretofore been used and is described, for example, in U.S. Pat. No. 5,733,739. Dilute acetic acid is known to “whiten” abnormal epithelium such as dermal papillomas (warts) and cervical lesions, and 5% acetic acid (vinegar) is routinely used in colposcopic examination. 5% acetic acid is also used to localize warts prior to treatment. The acetic acid “whitens” the tissue by condensing the nucleic acids (chromatin) within the nucleus. The condensed chromatin enhances the optical backscatter from the modified nucleus.